title: Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination.
abstract: A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Planchon
authors.forename: Liang
authors.initials: L
authors.lastname: Gao
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Milkie
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: James A
authors.initials: JA
authors.lastname: Galbraith
authors.forename: Catherine G
authors.initials: CG
authors.lastname: Galbraith
authors.forename: Eric
authors.initials: E
authors.lastname: Betzig
mesh_terms: D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D008938:Mitosis; D055620:Optical Phenomena; D013552:Swine
chemical_list: D008164:Luminescent Proteins